The KT-11 strain (GenBank accession number: AP025162, FERM BP-11332) was provided as a stock culture from KITII Co., Ltd. (Tokyo, Japan). KT-11 was inoculated in DeMan–Rogosa–Sharpe (MRS) broth, cultivated for 24 h at 37°C, collected by centrifugation, washed thrice with sterile water, and lyophilized.

Human rotavirus strains DS-1 (VR-2550) and Wa (VR-2018) were purchased from the American Type Culture Collection (Manassas, VA, United States). The viruses were treated with trypsin for 1 h before use, propagated in MA104 cells, and harvested after two freeze–thaw cycles.

The human colon carcinoma cell line Caco-2 was purchased from the Riken BioResource Research Center (Ibaraki, Japan). Cells were cultured in monolayer cultures using complete high-glucose Eagle’s minimal essential medium (EMEM; FUJIFILM Wako Pure Chemical, Osaka, Japan) containing 10% (v/v) heat-inactivated fetal bovine serum (FBS; Sigma-Aldrich, St. Louis, MO, United States), 100 U/mL penicillin G, and 100 μg/mL streptomycin (Sigma-Aldrich). The cells were passaged using TrypLE Express (Thermo Fisher Scientific, Waltham, MA, United States) and cultured under a humidified atmosphere of 5% CO₂/95% air at 37°C.

The KT-11 strain grown in MRS medium was collected by centrifugation (8,000 × g) at 4°C for 5 min. The pellet was washed by centrifugation (8,000 × g) thrice with cold distilled water. Bacterial cell pellets were suspended in 100 mL of 5 M lithium chloride and incubated in a shaker at 37°C for 2 h. Thereafter, the suspension was centrifuged (8,000 × g) at 4°C for 5 min, and the supernatant was filtered through a membrane filter with 0.20 μm pore diameter (Merck Millipore, Darmstadt, Germany). The filtrate was then dialyzed against distilled water at 4°C for 24 h. The dialyzate was centrifuged at 14,000 × g at 4°C for 5 min, and the precipitate was lyophilized to produce KT-11 lithium chloride extract (KT-11 LE) powder. KT-11 LE was obtained at a yield of 7.8%, determined on the basis of wet weight. KT-11 SLP concentrate (KT-11 SLP) was prepared from KT-11 LE using the method described by Johnson et al. (2013). Briefly, KT-11 LE was mixed with 1 M lithium chloride at 37°C for 24 h. The insoluble fraction was collected by centrifugation and lyophilized. KT-11 LE and KT-11 SLP was dissolved in sterile distilled water for subsequent study.

The effect of KT-11 strain and its components on rotavirus infectivity was assessed using the method shown in Figure 1. In pre-infection treatment, Caco-2 cells were seeded at 2.0 × 10⁵ cells/mL/well in 24-well plates (Thermo Fisher Scientific, Waltham, MA, United States) and cultured at 37°C. After 24 h, the culture supernatant was discarded, and the cells were incubated with the KT-11 LE or KT-11 SLP in 200 μL of serum-free EMEM for 1 h, following which either the Wa or the DS-1 strain was added and incubated for another 1 h. The culture supernatant was removed, the cells were washed with serum-free EMEM and further cultured in complete EMEM under a humidified atmosphere of 5% CO₂/95% air at 37°C. After 24 h, the cells were collected for quantitative reverse transcription-polymerase chain reaction (RT-PCR).

In post-infection treatment, Caco-2 cells were seeded at 2.0 × 10⁵ cells/mL/well in 24-well plates (Thermo Fisher Scientific) and cultured at 37°C. After 24 h, the culture supernatant was discarded, and Wa or DS-1 strain was added and incubated for another 1 h. Thereafter, the culture supernatant containing uninfected virus particles was removed, the cells were washed with serum-free EMEM and further cultured in complete EMEM supplemented with the KT-11 LE or KT-11 SLP under a humidified atmosphere of 5% CO₂/95% air at 37°C. After 24 h, the cells were collected for quantitative RT-PCR.

Total RNA was extracted from rotavirus-infected Caco-2 cells using TRI reagent (Sigma-Aldrich) according to the manufacturer’s protocol. The extracted RNA (1 μg) was reverse transcribed in a thermal cycler (PTC-200; MJ Research, Waltham, MA, United States) with 1 mM of each dNTP, 2.5 units/μL M-MLV reverse transcriptase (Thermo Fisher Scientific), and 10 pmol/μL of oligo(dT)18 primers at 42°C for 50 min. Quantitative RT-PCR of the resulting cDNA was performed using 0.5 μg cDNA with TB Green Premix Ex Taq II (Takara Bio, Shiga, Japan) and 10 pmol/μL primers. The primer sequences are shown in Table 1. The PCR comprised one cycle of preheating (95°C, 10 min) and 40 cycles of denaturation (95°C, 10 s) and primer annealing and extension (55°C, 30 s) using the Eco Real Time PCR System (Illumina, San Diego, CA, United States). Results were analyzed with the ΔΔCt method using the Eco system software (Illumina). The amount of PCR products was normalized to the expression level of GAPDH.

The identification of the expressed 45 kDa protein was based on the observed molecular weights of the proteins according to Antikainen et al. (2002). Briefly, the target band of the 45 kDa protein in 12% SDS-PAGE gel was digested by trypsin, followed by analysis with liquid chromatography-tandem mass spectrometry (LC-MS/MS). The generated data were subjected to a search using the Mascot (Matrix Science, London, United Kingdom) algorithm. Protein identification was carried out using all the SLP sequences of KT-11. Homology searches were performed using the BLAST program (available at¹). Sequences of eight Lactobacillus SLP sequences were aligned using CLUSTALW². The GenBank accession numbers for the SLP sequences were as follows: AAB58734 for CbsA of L. crispatus JCM 5810; AFB69875 for SlpA of L. crispatus K313; ABI49163 for SlpA of L. crispatus ZJ001: CAA61560 for SlpA of L. acidophilus JCM 4356; YP_193101 for SlpA from L. acidophilus NCFM; ATO53009 for the SLP of L. amylovorus JCM1126T; and CAB46985 for the SLP of L. helveticus JCM1120T. Both amino acid sequencing and peptide mapping were performed by Japan Proteomics Co., Ltd. (Miyagi, Japan).

Simulated digestive juice was prepared as gastric juice containing 1.6% pepsin (Sigma-Aldrich) in 0.1 M KCl/HCl (pH 2.0) and intestinal juice containing 0.1% pancreatin (Sigma-Aldrich) in PBS (pH 7.2). KT-11 LE and KT-11 SLP were treated with gastric juice or intestinal juice for the indicated time periods at 37°C. The sensitivity of KT-11 SLP to digestive enzyme treatment was evaluated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) using a discontinuous buffer system with 12.5% separating gel and 5% stacking gel. Protein bands were visualized by staining the gels with Quick-CBB (FUJIFILM Wako Pure Chemical).

To determine the inhibitory activity of SLP after gastric digestion, SLP was dissolved in gastric juice and incubated at 37°C. After treatment for 0, 0.5, 1, 2, and 3 h, the SLP solution was neutralized with NaOH to adjust pH to 7.2, and was subjected to the pre-infection treatment assay.

The data were statistically analyzed using one-way analysis of variance (ANOVA) with Tukey–Kramer multiple comparison tests using Excel 2019 (Microsoft, Redmond, WA, United States) with the add-in software Statcel4 (OMS Publishing, Tokyo, Japan). Results with a p-value of less than 0.05 were considered statistically significant.

The effect of KT-11 LE on the infection of DS-1 human rotavirus in Caco-2 cells is shown in Figures 2A,B. In the pre-infection treatment, KT-11 LE significantly suppressed the amplification of VP6 mRNA in a dose-dependent manner (Figure 2A). In contrast, in the post-infection treatment, no significant suppression of DS-1 strain infection was observed with KT-11 LE at all tested concentrations (Figure 2B).

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of KT-11 LE revealed the presence of 18, 30, 45, and 65 kDa proteins as its major proteinaceous components (Figure 3A). Among them, the 45 kDa protein was concentrated as an insoluble fraction in 1 M lithium chloride (Figure 3B).

The nine partial amino acid sequences of the 45 kDa peptide identified by LC-MS/MS were GTAVTDFSK, GSVNVTAGLNGR, LNVSTVASEILK, NFAAYQVSNGK, AVVADVNAALK, YNSVTVSPK, AYYEVVENGK, FINADNIDGTER, and VVTYGGTYTFK. Mascot search of these results revealed that all the sequences were included in the translated product of the SLP gene (LOCUS_02350) of KT-11 (Figure 3C). Comparing this amino acid sequence with SLPs from other Lactobacillus strains revealed that the 45 kDa protein was a novel SLP with significant homology in the C-terminal region and notable difference in the N-terminal and middle regions.

The effect of KT-11 SLP on the infection of DS-1 strain in Caco-2 cells is shown in Figure 4A. DS-1 infection was significantly suppressed by pre-infection treatment with KT-11 SLP in a concentration-dependent manner. Conversely, KT-11 SLP did not suppress the infection of the Wa strain even after pre-infection treatment at 100 μg/mL (Figure 4B).

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis patterns of KT-11 LE and the KT-11 SLP after exposure to simulated digestive juice are shown in Figures 5A,B. The 45 kDa protein band remained visible up to 3 h after the addition of simulated gastric juice, though it gradually disappeared with prolonged incubation. Only a slight decrease in the intensity of the 45 kDa protein band was observed till 6 h after the addition of simulated intestinal juice, while a more substantial decrease was observed after 24 h. Conversely, the bands of 18, 30, and 65 kDa proteins in KT-11 LE completely disappeared following the 24-h treatment with intestinal juice.

From 0 to 2 h after treatment with gastric juice, the inhibitory effect of KT-11 SLP on DS-1 strain infection in the pre-infection treatment decreased in a treatment time dependent manner. Significant inhibition was observed even after 2 h, but the activity was completely lost after 3 h digestion of KT-11 SLP with gastric juice (Figure 5C).